PROJECT SUMMARY Intrinsic immunity to HIV is counteracted by accessory virulence factors, which, typically, reprogram E3 Ub ligases to remove HIV restriction factors from infected cells by targeting them for degradation by proteasome. Indeed, the studies of HIV accessory proteins have led to the identification of major HIV restriction factors, which in turn revealed new anti-viral mechanisms. HIV-1 Vpr accessory protein binds to CRL4DCAF1 E3 ubiquitin ligase and uses this enzyme to antagonize specific postreplication DNA repair proteins, such as uracil DNA glycosylase (UNG2), HLTF DNA helicase and MUS81 structure specific nuclease. Furthermore, we recently identified Exonuclease 1 (Exo1) DNA repair protein as a novel target of the hijacked by Vpr CRL4DCAF1 E3. HIV-1 Vpr was also reported to bind DNA processing proteins, which associate with a large protein complex assembled on SLX4 scaffold that connects to diverse DNA repair pathways. The fact that Vpr interacts with and disrupts multiple aspects of the cellular DNA repair machinery is intriguing, as there is little doubt that Vpr antagonism with this machinery should ultimately benefit HIV-1. Nevertheless, very little is known about how the DNA repair proteins targeted by Vpr impinge on HIV-1 replication and the underlying mechanism(s) have not been thoroughly investigated. This application focuses on how DNA repair proteins antagonized by Vpr and those controlled by the SLX4 scaffold impinge on HIV-1 replication. We recently confirmed that HIV-1 Vpr promotes HIV-1 replication in dividing T cells and linked this effect to Vpr's ability to antagonize DNA repair proteins via CRL4DCAF1 E3. We also demonstrated that an as yet unidentified component controlled by the SLX4 scaffold inhibits HIV-1 infection. We hypothesize that the above DNA repair proteins act on intermediates in the synthesis of the double stranded HIV-1 cDNA prior to, or following provirus integration, thereby inhibiting HIV-1 infection, and that some of these effects are counteracted by HIV-1 Vpr. Here we propose to characterize the interactions between DNA repair proteins targeted by Vpr, as well as those controlled by the SLX4 scaffold, with the HIV-1 cDNA and elucidate how they inhibit HIV-1 replication. In particular, we will characterize the actions of DNA repair proteins that are antagonized by Vpr to stimulate HIV-1 replication (SA1), identify steps that the SLX4 complex and Vpr-CRL4DCAF1 E3-targeted HLTF and Exo1 act on (SA2), and characterize DNA repair pathways and activities that inhibit HIV-1 (SA3). Overall, this application will describe the molecular mechanisms mediating the inhibition of HIV-1 replication in dividing T lymphocytes by cellular DNA repair machinery, and those used by HIV-1 Vpr to antagonize the inhibition.